Are different forms of breathing derived from one or multiple neural networks? We demonstrate that brainstem slices containing the pre-Bötzinger complex generated two rhythms when normally oxygenated, with striking similarities to eupneic ('normal') respiration and sighs. Sighs were triggered by eupneic bursts under control conditions, but not in the presence of strychnine (1 microM). Although all neurons received synaptic inputs during both activities, the calcium channel blocker cadmium (4 microM) selectively abolished sighs. In anoxia, sighs ceased, and eupneic activity was reconfigured into gasping, which like eupnea was insensitive to 4 microM cadmium. This reconfiguration was accompanied by suppression of synaptic inhibition. We conclude that a single medullary network underlies multiple breathing patterns.

Neurons of the cerebellar nuclei have basal firing rates of 10-20 Hz, despite the convergence of many GABAergic Purkinje terminals onto cerebellar nuclear somata and the high spontaneous firing rate of Purkinje neurons. This persistence of firing during a constant barrage of inhibition raises the question of what patterns of Purkinje cell input inhibit nuclear cells most effectively. To explore the hypothesis that synaptic depression moderates inhibition at this synapse, we made whole-cell recordings from cerebellar nuclear neurons in mouse brain slices. IPSCs and IPSPs were elicited by electrically stimulating the corticonuclear tract at 10, 50, and 100 Hz. IPSCs evoked at the mean spontaneous firing rate of Purkinje cells (50 Hz) depressed by approximately 60%. The onset of depression had a fast, frequency-dependent component, from which recovery was rapid (approximately 100 msec), and a slower, frequency-independent component, from which recovery was slow (approximately 10 sec). As stimulation rate increased, steady-state depression increased, but each IPSC decayed less completely between stimuli, producing a "tonic" IPSC. Changes in stimulation rate produced rapid changes in the level of depression. Under current clamp, cerebellar nuclear neurons fired spontaneously. During 50 Hz trains of IPSPs, firing was initially interrupted, but resumed coincident with the onset of depression. Low-frequency trains entrained postsynaptic firing, and high-frequency trains greatly slowed firing, primarily because of the tonic IPSC. Thus, the properties of depression at this synapse appear to limit the sensitivity of nuclear cells to basal inhibition, while allowing the cells to respond to increases and decreases in Purkinje cell activity.

Cerebellar Purkinje neurons maintain high firing rates but their synaptic terminals depress only moderately, raising the question of how vesicle depletion is minimized. To identify mechanisms that limit synaptic depression, we evoked 100 Hz trains of GABAergic inhibitory postsynaptic currents (IPSCs) in cerebellar nuclear neurons by stimulating Purkinje axons in mouse brain slices. The paired-pulse ratio (IPSC(2)/IPSC(1)) of the total IPSC was approximately 1 and the steady-state ratio (IPSC(20)/IPSC(1)) was approximately 0.5, suggesting a high response probability of postsynaptic receptors, without an unusually high release probability. Three-dimensional electron microscopic reconstructions of Purkinje boutons revealed multiple active zones without intervening transporters, suggestive of "spillover"-mediated transmission. Simulations of boutons with 10-16 release sites, in which transmitter from any site can reach all receptors opposite the bouton, replicated multiple-pulse depression during normal, high, and low presynaptic Ca influx. These results suggest that release from multiple-site boutons limits depletion-based depression, permitting prolonged, high-frequency inhibition at corticonuclear synapses.

We examined the role of the hyperpolarization-activated current (I(h)) in the generation of the respiratory rhythm using a spontaneously active brainstem slice of mice. This preparation contains the hypoglossus (XII) nucleus, which is activated in-phase with inspiration and the pre-Bötzinger complex (PBC), the presumed site for respiratory rhythm generation. Voltage-clamp recordings (n = 90) indicate that cesium (Cs) (5 mM) blocked 77.2% of the I(h) current, and ZD 7288 (100 microM) blocked 85.8% of the I(h) current. This blockade increased the respiratory frequency by 161% in Cs and by 150% in ZD 7288 and increased the amplitude of integrated population activity in the XII by 97% in Cs and by 162% in ZD 7288, but not in the PBC (Cs, by 19%; ZD 7288, by -4.56%). All inspiratory PBC neurons (n = 44) recorded in current clamp within the active network revealed a significantly decreased frequency of action potentials during the interburst interval and an earlier onset of inspiratory bursts after I(h) current blockade. However, hyperpolarizing current pulses evoked only in a small proportion of inspiratory neurons (0% of type I; 29% of type II neurons) a depolarizing sag. Most of the neurons expressing an I(h) current (86%) were pacemaker neurons, which continued to generate rhythmic bursts after inactivating the respiratory network pharmacologically with CNQX alone or with CNQX, AP-5, strychnine, bicuculline, and carbenoxolone. Cs and ZD 7288 increased the frequency of pacemaker bursts and decreased the frequency of action potentials between pacemaker bursts. Our findings suggest that the I(h) current plays an important role in modulating respiratory frequency, which is presumably mediated by pacemaker neurons.

No abstract